<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> aggravates vein graft intimal hyperplasia in a rat model

Background  

Along with angioplasty, autologus vein grafts are commonly used for artery bypass grafting in patients with advanced arterial stenosis and drug-resistant angina pectoris. Although initially a successful procedure, long-term functionality is limited due to proliferation and migration of smooth muscle cells. Like in atherosclerosis, common chronic infections caused by viruses and bacteria may contribute to this process of vein graft failure. Here we investigated the possible role of Chlamydia pneumoniae (Cpn) in the pathogenesis of venous graft failure in an experimental animal model. In 2 groups (n = 10 rats/group), an epigastric vein-to-common femoral artery interposition graft was placed. Immediately thereafter, rats were infected with Cpn (5*10⁸ IFU) or injected with control solutions. Rats were sacrificed three weeks after surgery and the grafts were harvested for morphometrical and immunohistochemical analysis.     

静脉桥狭窄家兔动物模型的建立

目的建立冠心病冠状动脉旁路移植术后移植静脉桥狭窄的动物模型。方法取3.0～3.5 kg普通新西兰兔8只,取同侧颈外静脉与颈总动脉进行端端吻合,吻合时采用间断缝合的方法。结果术后2周、4周取下静脉桥及对侧颈外静脉,光镜下见静脉桥新生内膜形成,中膜增厚,弹力纤维减少;胶原纤维不均匀性增厚。结论本模型能反映冠状动脉旁路移植术后静脉桥狭窄的情况,可满意模拟人冠状动脉旁路移植术后大隐静脉桥的病理变化。     

A simple fluid-structure coupling algorithm for the study of the anastomotic mechanics of vascular grafts

Vascular anastomoses constitute a main factor in poor graft performance due to mismatches in distensibility between the host artery and the graft. This work aims at computational fluid-structure investigations of proximal and distal anastomoses of vein grafts and synthetic grafts. Finite element and finite volume models were developed and coupled with a user-defined algorithm. Emphasis was placed on the simplicity of the coupling algorithm. An artery and vein graft showed a larger dilation mismatch than an artery and synthetic graft. The vein graft distended nearly twice as much as the artery while the synthetic graft displayed only approximately half the arterial dilation. For the vein graft, luminal mismatching was aggravated by development of an anastomotic pseudo-stenosis. While this study focused on end-to-end anastomoses as a vehicle for developing the coupling algorithm, it may serve as useful point of departure for further investigations such as other anastomotic configurations, refined modelling of sutures and fully transient behaviour.     

Beobachtungen über das Befalls-“decline„-Phänomen beim Auftreten von Fußkrankheiten in Monokulturen

In comparative experiments for detecting apple chlorotic leaf spot virus (ApCLSV) in apple cultivars and wild apple varieties using graft indexing the procedure of applying the woody indicator clone Malus pumila MILL. Comparing R 12740 7A and the DAS‐ELISA technique, the reliability of the ELISA in ApCLSV detection in fruit‐bearing cultivars proved to be slightly better than that of R 12740 7A. In certain taxa of wild apple varieties, however, the reliability of the graft indexing procedure in detecting ApCLSV proved to be much better than that of ELISA. The investigation was carried out with 864 trees belonging to 16 apple cultivars and 24 wild apple varieties, respectively.     

The clopidogrel after surgery for coronary artery disease (CASCADE) randomized controlled trial: clopidogrel and aspirin versus aspirin alone after coronary bypass surgery [NCT00228423]

Background

Saphenous vein graft disease remains a major limitation of coronary artery bypass graft surgery. The process of saphenous vein intimal hyperplasia begins just days after surgical revascularization, setting the stage for graft atherosclerotic disease and its sequalae. Clopidogrel improves outcomes in patients with atherosclerotic disease, and is effective at reducing intimal hyperplasia in animal models of thrombosis. Therefore, the goal of this study will be to evaluate the efficacy of clopidogrel and aspirin therapy versus aspirin alone in the prevention of saphenous vein graft intimal hyperplasia following coronary artery bypass surgery.

Methods

Patients undergoing multi-vessel coronary artery bypass grafting and in whom at least two saphenous vein grafts will be used are eligible for the study. Patients will be randomized to receive daily clopidogrel 75 mg or placebo, in addition to daily aspirin 162 mg, for a one year duration starting on the day of surgery (as soon as postoperative bleeding has been excluded). At the end of one year, all patients will undergo coronary angiography and intravascular ultrasound assessment of one saphenous vein graft as selected by randomization. The trial will be powered to test the hypothesis that clopidogrel and aspirin will reduce vein graft intimal hyperplasia by 20% compared to aspirin alone at one year following bypass surgery.

Discussion

This trial is the first prospective human study that will address the question of whether clopidogrel therapy improves outcomes and reduces saphenous vein graft intimal hyperplasia following cardiac surgery. Should the combination of clopidogrel and aspirin reduce the process of vein graft intimal hyperplasia, the results of this study will help redefine modern antiplatelet management of coronary artery bypass patients.     

Enhancement of nerve regeneration and orientation across a gap with a nerve graft within a vein conduit graft: a functional, stereological, and electrophysiological study

In this study, the right sciatic nerves of 40 rats were used to determine whether a nerve graft within a vein graft might accelerate and facilitate axonal regeneration, compared with a nerve graft alone. The animals were separated into four groups, as follows: group 1, sham control; group 2 (control), segmental nerve resection and no repair; group 3, segmental nerve resection and nerve grafting; group 4, segmental nerve resection and reconstruction with a nerve graft within a vein conduit graft. For all groups, sciatic functional indices were calculated before the operation and on postoperative days 7 and 90. On postoperative day 90, the sciatic nerves were reexposed and nerve conduction velocities were recorded. The sciatic nerves were harvested from all groups for counting of the myelinated axons with a stereological method. No statistically significant differences with respect to return of gait function, axon count, or nerve conduction were noted between groups 3 and 4 (p > 0.05). However, functional recovery in group 4 on postoperative day 90 was significant, compared with group 2 (p < 0.05); the recovery difference between groups 2 and 3 was not significant (p > 0.05). This study was not able to demonstrate any functional benefits with the use of a nerve graft within a vein graft, compared with standard nerve grafting.     

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.^1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. ^6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.^9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., ⁹ as well as others.^12-14 Nevertheless, these cited small animal protocols are complicated.To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8±3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.     

Ex vivo electroporation as a potent new strategy for nonviral gene transfer into autologous vein grafts

Gene transfer to vein grafts has therapeutic potential to prevent late graft failure; however, certain issues, including efficacy and safety, have hindered the clinical application of this treatment modality. Here, we report the successful and efficient gene transfer of plasmid DNA via ex vivo electroporation into veins as well as into vein grafts. Two approaches were used: one involved transluminal in situ gene transfer using a T-shaped electrode (the "Lu" method), and the other was an adventitial ex vivo approach using an electroporation cuvette followed by vein grafting (the "Ad" method). The Lu method was carried out at 10 V, with optimal gene transfer efficiency in the in situ jugular veins of rabbits, and transgene expression was observed primarily in endothelial cells. However, when these veins were grafted into the arterial circulation, no luciferase activity was detected; this effect was probably due to the elimination of the gene-transferred cells as a result of endothelial denudation. In contrast, optimal and satisfactory gene transfer was obtained with the vein grafts subjected to the Ad method at 30 V, and transgene expression was seen primarily in adventitial fibroblasts. Gene transfer of endothelial nitric oxide synthase cDNA to the vein graft via the Ad method successfully limited the extent of intimal hyperplasia, even under hyperlipidemic conditions, at 4 wk after grafting. We thus propose that the Ad method via ex vivo electroporation may provide a novel, safe, and clinically available technique for nonviral gene transfer to sufficiently prevent late graft failure.     

Computational model of blood flow in the aorto-coronary bypass graft

Background

Coronary artery bypass grafting surgery is an effective treatment modality for patients with severe coronary artery disease. The conduits used during the surgery include both the arterial and venous conduits. Long- term graft patency rate for the internal mammary arterial graft is superior, but the same is not true for the saphenous vein grafts. At 10 years, more than 50% of the vein grafts would have occluded and many of them are diseased. Why do the saphenous vein grafts fail the test of time? Many causes have been proposed for saphenous graft failure. Some are non-modifiable and the rest are modifiable. Non-modifiable causes include different histological structure of the vein compared to artery, size disparity between coronary artery and saphenous vein. However, researches are more interested in the modifiable causes, such as graft flow dynamics and wall shear stress distribution at the anastomotic sites. Formation of intimal hyperplasia at the anastomotic junction has been implicated as the root cause of long- term graft failure.Many researchers have analyzed the complex flow patterns in the distal sapheno-coronary anastomotic region, using various simulated model in an attempt to explain the site of preferential intimal hyperplasia based on the flow disturbances and differential wall stress distribution. In this paper, the geometrical bypass models (aorto-left coronary bypass graft model and aorto-right coronary bypass graft model) are based on real-life situations. In our models, the dimensions of the aorta, saphenous vein and the coronary artery simulate the actual dimensions at surgery. Both the proximal and distal anastomoses are considered at the same time, and we also take into the consideration the cross-sectional shape change of the venous conduit from circular to elliptical. Contrary to previous works, we have carried out computational fluid dynamics (CFD) study in the entire aorta-graft-perfused artery domain. The results reported here focus on (i) the complex flow patterns both at the proximal and distal anastomotic sites, and (ii) the wall shear stress distribution, which is an important factor that contributes to graft patency.

Methods

The three-dimensional coronary bypass models of the aorto-right coronary bypass and the aorto-left coronary bypass systems are constructed using computational fluid-dynamics software (Fluent 6.0.1). To have a better understanding of the flow dynamics at specific time instants of the cardiac cycle, quasi-steady flow simulations are performed, using a finite-volume approach. The data input to the models are the physiological measurements of flow-rates at (i) the aortic entrance, (ii) the ascending aorta, (iii) the left coronary artery, and (iv) the right coronary artery.

Results

The flow field and the wall shear stress are calculated throughout the cycle, but reported in this paper at two different instants of the cardiac cycle, one at the onset of ejection and the other during mid-diastole for both the right and left aorto-coronary bypass graft models. Plots of velocity-vector and the wall shear stress distributions are displayed in the aorto-graft-coronary arterial flow-field domain. We have shown (i) how the blocked coronary artery is being perfused in systole and diastole, (ii) the flow patterns at the two anastomotic junctions, proximal and distal anastomotic sites, and (iii) the shear stress distributions and their associations with arterial disease.

Conclusion

The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear-stress variation is observed around the distal anastomotic region. These results can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease.

     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

The venous skin graft method for repairing skin defects of the fingers

A venous skin graft for the treatment of skin defects in a finger is described. This procedure involves taking a flap from the forearm together with the subcutaneous vein and anastomosing both ends of this vein to the digital artery and vein, respectively. Thirteen difficult finger wounds were resurfaced with such a venous skin graft. The sizes of the flaps ranged from 1.3 X 3.0 cm to 2 X 5 cm. The lengths of the veins taken were from 6 to 12 cm. Subcutaneous fat is thin, and there is good elasticity in the grafted flap.     

Towards the treatment of saphenous vein bypass graft failure--a perspective of the Bristol Heart Institute

Coronary artery bypass graft surgery (CABG) is widely used for the treatment of atheromatous stenosis of coronary arteries. However, as many as 50% of grafts fail within 10 years after CABG due to neointima (NI) formation, a process involving the proliferation and migration of vascular smooth muscle cells (VSMCs). Superimposed on neointima formation is accelerated atherogenesis which ultimately results in late vein graft failure. To date no therapeutic intervention has proved successful in treating late vein graft failure and as such is a matter of some urgency. However, in recent years, several diverse approaches aimed at preventing neointimal formation have been devised which have yielded promising results. These include the use of external stents, gene therapy as well as conventional pharmacological interventions. The objective of this article, therefore, is to review these recent approaches and their potential clinical applications in the treatment of vein graft disease.     

Early detection of grapevine leafroll virus in <Emphasis Type="Italic">Vitis vinifera</Emphasis> using <Emphasis Type="Italic">in vitro</Emphasis> micrografting

Micrografting of grapevine was investigated for its use as a tool in virus indexing of grapevine stock. Cabernet franc and Cabernet sauvignon scions infected with grapevine leafroll-associated closterovirus III (GLRaVIII) were grafted on to virus-free indicator rootstocks of LN 33 and Cabernet sauvignon growing in tissue culture. The two rootstocks and two scions were grafted in all four possible combinations along with two control grafts (virus-free scion on virus-free rootstock). A modified MS Murashige and Skoog (1962) tissue culture medium supplemented with 0.5 mg l^–1 6-benzylaminopurine was sufficient to induce multiple shoots. Shoots and micrografts readily produced roots in the basal medium. Micrografting gave an overall success rate of 77.8%, with no significant difference between LN 33 rootstock and Cabernet sauvignon. When leafroll infected scion material was micrografted on to virus-free rootstock, the rootstock leaf turned red (23.5% in LN 33 and 63.9% in Cabernet sauvignon) or it showed leafrolling (28.5%, no significant difference between LN 33 and Cabernet sauvignon) within 2–3 weeks. After 12 weeks in culture, the extent of viral symptoms in the micrografted material was high (81.3%), with no significant difference between LN 33 and Cabernet sauvignon; however, the expression of symptoms was more severe on Cabernet sauvignon than on LN 33 rootstock. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) was used to validate the visual symptoms and the presence of virus was confirmed in 80% of the rootstock with visual symptoms of infection. Results indicate that micrografting is an effective method for viral indexing of grapevines. The method can be used in conjunction with wood indexing for post-entry quarantine to identify infected material and reject it much earlier than is currently possible.     

Predictive Factors of Late Venous Aortocoronary Graft Failure: Ultrastructural Studies

Background

Venous aortocoronary graft arterialization may precede a preterm occlusion in some coronary artery bypass grafting (CABG) patients. The aim of the present study was to identify ultrastructural variations in the saphenous vein wall that may have an impact on the development of venous graft disease in CABG patients.

Methods

The study involved 365 consecutive patients with a mean age of 62.9±9.4 years who underwent isolated CABG. The thickness and area of the whole venous wall, the tunica intima, the tunica media and the adventitia and the number and shape (length, thickness and length/thickness ratio) of the nuclei in the medial smooth muscle cells nuclei in the distal saphenous vein segments were evaluated by ultrastructural studies. Patients were followed up for 41 to 50 months (mean 45.1±5.1). Saphenous vein graft patency was assessed by follow-up coronary angiography. Logistic regression models were used to identify independent risk factors for late graft failure.

Results

In 71 patients significant lesions in the saphenous vein grafts were observed. The whole venous wall thickness (437.5 µm vs. 405.5 µm), tunica media thickness (257.2 µm vs. 211.5 µm), whole venous wall area (2.23 mm² vs. 2.02 mm²) and tunica media area (1.09 mm² vs. 0.93 mm²) were significantly larger for this group of patients than for those without graft disease. In the latter group more elongated smooth muscle cell nuclei (higher length/thickness ratio) were found in the tunica media of the saphenous vein segments. Thickening of the saphenous vein tunica media and chunky smooth muscle cell nuclei were identified as independent risk factors for graft disease development.

Conclusions

Saphenous vein tunica media hypertrophy (resulting in wall thickening) and chunky smooth muscle cell nuclei might predict the development of venous graft disease.     

Characterization of the Inhibition of Vein Graft Intimal Hyperplasia by a Biodegradable Vascular Stent

Characterization of biodegradable stent vein graft thickening. Polydioxanone vascular sutures (PDSs) were used in a biodegradable arteriovenous bypass model. Twenty-four rabbits underwent carotid interposition bypass via ipsilateral jugular vein. One half received the stent (PDS group) and the remaining half a simple vein graft (controls). Group subsets received external stent removal or sham-control exploration at 4 and 12 weeks. At 4 and 12 weeks, the PDS group had significantly less medial and intimal thickening than the control group (P < 0.05), and there were fewer proliferating smooth muscle cells and extra cellular matrix formation than the control group at every interval. At 12 weeks, partial stent degradation occurred without deleterious effects. Furthermore proliferating cell nuclear antigen (PCNA), angiotensin type 1 receptor (AT1R), and transforming growth factor beta 1 (TGF-β1) levels were significantly lower than the control group. The external stent inhibited medial and intimal hyperplasia, an effect that remains after the material has completely degraded. This PDS stent is feasible option for vein grafts.     

Another method to prevent venous thrombosis in microsurgery: an in situ venous catheter

Free-flap failure is in the order of 4 to 10 percent. Heparin is more effective at preventing venous thrombosis than arterial thrombosis. This study was undertaken to investigate the efficacy of delivering heparin at a high dose locally but low dose systemically (heparin infusion via a catheter placed proximal to the venous anastomosis) to prevent venous thrombosis in microsurgery. A model of venous thrombosis was first established by a venous inversion graft in the rat femoral vein (this was performed in seven animals and resulted in 100 percent thrombosis). Saline and heparin were delivered proximal to the inverted vein graft to assess the effect of each in preventing venous thrombosis. Flow/patency distal to the inverted vein graft was assessed by observation under the microscope, the milk test, and rate of flow (flowmeter). Saline infused via a catheter proximal to the venous inversion graft resulted in 100 percent thrombosis in 10 animals. Heparin (100 U/ml at 2 to 3 ml/hour) infused through a catheter for 2 hours proximal to the anastomosis resulted in flow in all 10 animals during the infusion. Blood was also taken before beginning the procedure (control) and after the heparin infusion distal to the anastomosis (local partial thromboplastin time) as well as in the contralateral femoral vein (systemic). The control for all animals that received heparin was <3 minutes. The systemic partial thromboplastin time after heparin infusion was <3 minutes in seven animals, 3.3 minutes in two animals, and >7 minutes in one animal. The local partial thromboplastin time distal to the inverted vein graft was >10 minutes in nine animals and 3.7 minutes in one animal. The study also had a clinical component, in which a catheter was placed in a vein of the free flap, and heparin was infused over 5 days. This technique has been used in 83 consecutive free flaps. In three recent free flaps performed on the limbs, the local partial thromboplastin time (close to the anastomosis) was raised but the systemic time was normal. This technique offers a method in preventing venous thrombosis in microsurgery. It is simple to implement and is not associated with the systemic complications of heparin.     

The role of cell proliferation and migration in the development of a neo-intimal layer in veins grafted into arteries,in rats

Summary The development of a thickened (hyperplastic) fibro-cellular neo-intima is a significant event in the adaptation of a vein grafted into an artery. The histogenesis of tissues in vein grafts was explored in a rat model where the source of endothelial and smooth muscle cells was from the adjacent artery. Cell proliferation was assessed by the incorporation of tritiated thymidine and autoradiography, up to 18 months after grafting. Cell migration was detected by prelabelling in the first 5 days after grafting and sampling at later times. The proliferation of cells in the arterial media adjacent to the graft was elevated above control levels as early as 2 days after grafting; it was maximal at 3 days and returned to low levels by day 21. During the first week, prelabelled smooth muscle cells in the tunica media of the adjacent artery migrated to the subendothelial space, where they continued to proliferate to produce arterial intimal hyperplasia. The migration of endothelial and smooth muscle cells proceeded across the anastomosis to populate the vein graft neo-intima, where smooth muscle cells continued to proliferate until 28 days after grafting. Cell migration and proliferation were significant factors in the histogenesis of vein graft neo-intimal hyperplasia in this model. These processes were controlled, perhaps by local regulatory factors, to form a vein graft, the wall of which was similar in thickness and structure to that of the host artery.